The field of this invention is the production of large size lipid vesicles or liposomes. In particular, the invention is concerned with the production of giant size liposomes from small unilamellar vesicles (SUV) by a freeze-thaw manipulation of the SUV aqueous dispersions.
Processes have heretofore been described for producing larger sizes of liposomes in which SUV produced by sonication are induced to enlarge by fusion in the presence of calcium ions, or by techniques utilizing organic solvents or detergents. The calcium-fusion method is limited to certain lipids. The production of large unilamellar liposomes by using organic solvents or detergents has its limitations for entrapping biological agents, since such agents may be activated by the solvent or detergent.
In 1977, Kasahara and Hinkel described a procedure which produced enlargement of liposomes, consisting of an initial sonication followed by freezing, thawing, and a further brief sonication (J. Biol. Chem. 252: 7384-7390). Subsequently, Pick described further experiments in which liposomes with a large trapping capacity were prepared by freezing and thawing of sonicated phospholipid mixtures. Arch. Biochem. Biophys. (1981) 212: 186-194. Pick estimated that the maximal trapping efficiency of the procedure was about 25-30% of the ambient solution, and further found that a number of conditions inhibited enlargement and maximized trapping. These included increasing the ionic strength of the medium and/or increasing the liposome concentration. Pick was also unable to obtain satisfactory enlargement of the vesicles using purified egg phospholipid (phosphatidylcholine).